Detection and characterization of microorganisms

ABSTRACT

A method for separating microorganisms, especially infectious agents, from a mixture by two dimensional centrifugation on the basis of sedimentation rate and isopycnic banding density, for sedimenting such microorganisms through zones of immobilized reagents to which they are resistant, for detecting banded particles by light scatter or fluorescence using nucleic acid specific dyes, and for recovering the banded particles in very small volumes for characterization by mass spectrometry of viral protein subunits and intact viral particles, and by fluorescence flow cytometric determination of both nucleic acid mass and the masses of fragments produced by restriction enzymes. The method is based on the discovery that individual microorganisms, such as bacterial and viral species, are each physically relatively homogeneous, and are distinguishable in their biophysical properties from other biological particles, and from non-biological particles found in nature. The method is useful for distinguishing infections, for identifying known microorganisms, and for discovering and characterizing new microorganisms. The method provides very rapid identification of microorganisms, and hence allows a rational choice of therapy for identified infectious agents. A particularly useful application is in clinical trials of new antibiotics and antivirals, where it is essential to identify at the outset individuals infected with the targeted infectious agent.

CROSS-REFERENCE TO RELATED APPLICATION

[0001] The present application is a division application of U.S. patentapplication Ser. No. 09/571,279 filed May 16, 2000, which is adivisional of U.S. patent application Ser. No. 09/265,541, filed Mar. 9,1999, each incorporated herein by reference. The present application isfurther related to U.S. provisional patent application Serial No.60/077,472, filed Mar. 10, 1998, incorporated herein by reference, andclaims priority thereto under 35 USC §119(e).

[0002] This invention was made with Government support under an SBIRgrants from NIH, Grant Nos. 1 R43 AI41819-01/02. The United Statesgovernment may have certain rights in the invention.

BACKGROUND OF THE INVENTION

[0003] The present invention relates to the field of separating andidentifying microorganisms, particularly infectious agents, usingtwo-dimensional centrifugation and exposure to chemical and enzymaticagents, combined with detection in density gradients based on lightscatter or fluorescence, counting by fluorescence flow cytometry, andcharacterization of intact virions, bacteria, proteins and nucleic acidsby mass spectrometry, flow cytometry and epifluorescence microscopy.

[0004] The publications and other materials used herein to illuminatethe background of the invention or provide additional details respectingthe practice, are incorporated by reference, and for convenience arerespectively grouped in the appended List of References. Patentsreferenced herein are also incorporated by reference.

[0005] In the prior art, diagnosis of viral and bacterial infections hasbeen done by culturing the causal agents in suitable media or in tissueculture to obtain sufficient particles for analysis, followed byidentification based on which conditions support growth, on reaction tospecific antibodies, or based on nucleic acid hybridization (Gao andMoore, 1996). Biological growth can be omitted when the polymerase chainreaction (PCR) is used to amplify DNA, however, PCR requiressequence-specific primers, and is thus limited to known or suspectedagents (Bai et al., 1997). For all these methods, considerable time isrequired, and the methods are useful for agents whose properties areknown or suspected. Existing methods do not provide means for rapidlyisolating and characterizing new infectious agents. Hundreds ofinfectious agents are known, and it is infeasible to have availablereagents for an appreciable fraction of them.

[0006] Techniques for recovering infectious agents from blood, urine,and tissues have been previously developed based on centrifugation orfiltration, but have not been widely used clinically (Anderson et al.,1966; Anderson et al., 1967). The highest resolution methods use ratezonal centrifugation to separate fractions based on sedimentation rate(measured in Svedberg units, S) and isopycnic banding density (measuredin grams per mL or ρ). S-ρ separations have been used to isolate virusparticles in a high state of purity from rat liver homogenates, and havebeen used to isolate the equivalent of approximately 20 virions per cell(Anderson et al., 1966). In these studies, virus particles were detectedby light scattering and visualized by electron microscopy. Theseparations required complex special equipment not generally available,one or more days of effort, and they did not provide a definitiveidentification of the bacterial or viral species separated.

[0007] It is important to show that candidate infectious particlesisolated by centrifugal methods actually contain nucleic acids. DNA andRNA in both active and fixed bacterial and viral particles have beenstained with fluorescent dyes specific to nucleic acids, and observedand counted by fluorescent microscopy and flow cytometry. Many dyes arenow known which exhibit little fluorescence in the free state, butbecome highly fluorescent when bound to nucleic acids. Some binddifferentially to DNA or RNA or to different specific regions, and someshow different emission spectra depending on whether bound to DNA orRNA. In this disclosure, dyes referred to are fluorescent dyes. Bydifferential fluorescence spectroscopy ssDNA, dsDNA and RNA may bedistinguished. See, Haugland, 1996; Mayor and Diwan, 1961; Mayor, 1961;Hobbie et al., 1977; Zimmerman, 1977; Perter and Feig, 1980; Paul, 1982;Suttle, 1993; Hirons et al., 1994; Hennes and Suttle, 1995; Hennes etal., 1995.

[0008] Isolated nucleic acid molecules of the dimensions found inbacteria and viruses have been counted and their mass estimated usingfluorescence flow cytometry for molecules in solution, andepifluorescence microscopy of immobilized molecules (Hennes and Suttle,1995, Goodwin et al., 1993). In both instances, the size of fragmentsproduced by restriction enzymes can be estimated, and the moleculesidentified by reference to a database listing the sizes of fragments ofknown DNA molecules produced by different restriction enzymes (Hammondet al., U.S. Pat. No. 5,558,998; Jing et al., 1998).

[0009] Using specific fluorescently-labeled antibodies, specificidentifications may also be made. These studies are time consuming, andrequire batteries of specific antibodies, together with epifluorescentmicroscopy or fluorimeters.

[0010] Matrix-Assisted Laser-Desorption-Ionization Time-of-Flight MassSpectrometry (MALDI-TOF-MS) currently allows precise measurements of themasses of proteins having molecular weights of over 50,000 daltons.Individual virion proteins have been previously studied by massspectrometry (Siuzdak, 1998); however, resolution of complete sets ofviral subunits from clinically relevant preparations of intact viruses,and the demonstration that precise measurements could be made of theirindividual masses, have not been previously reported. While singleprotein mass measurements can reliably identify many proteins, when aset of proteins from a virus or bacterial cell are known, detection ofsuch a set provides more definitive identification. Methods arecurrently also being developed which allow partial sequencing ofproteins or enzymatically produced peptide fragments and thus furtherincrease the reliability of identifications. For MALDI-TOF-MS currentlyused methods require a picomole or more of protein, while electrospraymass spectrometry currently requires 5-10 femtomoles. The detectionlimits with mass spectrometry, especially MALDI, depend on getting asample concentrated and on to a very small target area. Sensitivity willincrease as ultramicro methods for concentrating and transferring eversmaller-volume samples are developed. See, Claydon et al., 1996;Fenselau, 1994; Krishmanurthy et al., 1996; Loo et al., 1997; Lennon andWalsh, 1997; Shevchenko et al., 1996; Holland et al., 1996; Liang etal., 1996.

[0011] Centrifugal methods for concentrating particles from large intosmall volumes have been in use for decades. Using microbandingcentrifuge tubes which have a large cylindrical volume and cross sectionwhich tapers gradually in a centrifugal direction down to a smalltubular section, particles may be concentrated or banded in a densitygradient restricted to the narrow tubular bottom of the tube, or may bepelleted. The basic design of such tubes are well known by those skilledin the arts. See, Tinkler and Challenger, 1917; Cross, 1928; ASTMCommittee D-2, 1951; Davis and Outenreath, U.S. Pat. No. 4,624,835;Kimura, U.S. Pat. No. 4,861,477; Levine et al., U.S. Pat. No. 5,342,790;Saunders et al., U.S. Pat. No. 5,422,018; Saunders, U.S. Pat. No.5,489,396. The original tubes of this type were called Sutherland bulbsand were used to determine the water content of petroleum (The Chemistryof Petroleum and Its Substitutes, 1917, ASTM Tentative Method of Testfor Water and Sediment by Means of Centrifuge, ASTM Designation: D96-50T, 1947). Slight modifications of the basic design are described inU.S. Pat. Nos. 4,106,907; 4,624,835; 4,861,477; 5,422,018, 5,489,396.Such tubes have been made of glass or plastic materials, and the use ofwater or other fluids to support glass or plastic centrifuge tubes inmetal centrifuge shields has long been well known in the art. However,centrifuge tubes disclosed in the prior art which include a shapesimilar to that of the microbanding centrifuge tubes of the instantinvention could not withstand the centrifugal forces required to bandviral particles in gradients. Conventional centrifuge tubes, or tubesderivative from the Sutherland design have been used for densitygradient separations, and for separations in which wax or plasticbarriers are used which position themselves between regions of differentdensity to allow recovery of these fractions without mixing. There hasbeen no previous discussion of barriers which prevent mixing of stepgradient components at rest, but which barriers are centrifuged awayfrom the gradient during rotation. Nor have tube closures for high-speedthin-walled swinging-bucket centrifuge tubes been described, whoseexterior surfaces can be disinfected after the tubes are loaded.

[0012] The efficient stabilization of very shallow density gradients incentrifugal fields is well known, and is utilized in analyticalultracentrifugation to cause a sample layer to flow rapidly to thecentripetal surface of a gradient without mixing using a syntheticboundary cell (Anderson, U.S. Pat. No. 3,519,400). Hence, light physicalbarrier disks between step gradient components can be moved away fromthe gradient by centrifugal force without appreciably disturbing thegradient, provided that they are made of porous, woven or sinteredmaterials having a physical density less than that of the sample layer,such as polyethylene or polypropylene.

[0013] Many authors have noted that viruses and bacteria are oftenresistant to the actions of detergents and enzymes which will digest ordissolve contaminating particles of biological origin, and efforts havebeen made to classify infectious agents on the basis of theirdifferential sensitivities. These differences have not previously andconveniently been incorporated in a method for detecting and quantifyinginfectious agents. See, Gessler et al., 1956; Theiler, 1957; Epstein andHold, 1958; Kovacs, 1962; Planterose et al., 1962; Gard and Maaloe,1959. Density differences between different species of virus andbacteria are well known, but have not been previously exploited forpurposes of identification.

[0014] Infectious particles exhibit a wide range of isopycnic bandingdensities ranging from approximately 1.17 g/ml to 1.55 g/ml, dependingon the type of nucleic acid present, and the ratios between the amountof nucleic acid, protein, carbohydrate, and lipid present. While suchbanding density differences are well known, no attempt has beenpreviously made to systematically measure them and use the data toclassify infectious agents.

[0015] The present invention is directed to an integrated system forconcentrating, detecting and characterizing infectious agents usingseparations based on sedimentation rate and banding density, spectralanalysis of emitted fluorescent light to distinguish DNA from RNA,differentiation of viral and bacterial particles from other particles bysedimentation through zones of solubilizing enzymes or reagents,determination of the isopycnic banding densities of infectious particlesby reference to the positions of synthetic density standardizationparticles, particle detection using fluorescent dyes for DNA or RNA,further concentration of banded particles by pelleting, transfer ofconcentrated particles to mass spectrometer targets for protein massdetermination and analysis, counting of concentrated particles byepifluorescent microscopy and fluorescence flow cytometry, andidentification of bacterial or viral nucleic acids by restrictionfragment length polymorphism s analysis using either immobilized nucleicacid molecules, or ultrasensitive fluorescence flow cytometry. Thesemethods are especially useful in characterizing biological samples whichhave low titres of virus and which contain viruses which are notculturable.

[0016] Furthermore, all current methods used to detect and characterizeinfectious agents, including use of fluorescent antibodies, detection ofagent-associated enzymes, culture to increase agent mass, PCRamplification, restriction fragment length polymorphism analysis,hybridization to probes immobilized on chips, histochemical analysis,and all forms of microscopy including electron microscopy, are vastlyimproved by preconcentration of the microorganisms using the methods ofthe present invention.

[0017] These techniques have not previously been assembled into oneoperational system capable of routine field, hospital, and clinicallaboratory use. The present application describes innovations andinventions which make such a system feasible. For work with potentiallylethal agents, the system will be assembled in containment, and at leastpartially automated.

SUMMARY OF THE INVENTION

[0018] The overall objective of this invention is to develop a physicalsystem for rapidly identifying infectious disease agents without growingthem, and for discovering new infectious agents The process is based onthe thesis that infectious agents constitute a unique group of particleswhich can be isolated by physical and chemical means from other naturalparticles and identified by their physical parameters using centrifugalmeans, fluorescence, and mass spectrometry. The system will allow rapidclinical distinction between viral and bacterial infections,identification of specific agents with the aim of providing specifictherapy, and the rapid discovery of new infectious agents. In additionthe system will make it feasible to develop and test new antibiotics andantiviral agents in man by measuring the effects of these agents onbacterial and viral loads. At present the development of new antiviraldrugs is severely hindered by inability to define populations ofindividuals in the early stages of infection who might benefit fromtreatment.

[0019] In accordance with the present invention, an ultracentrifuge tubeis provided which comprises upper, middle and lower regions ofsuccessively smaller diameters. In one embodiment, the tube has an upperregion for receiving a sample, a funnel-shaped middle region and a lowernarrow tubular microbanding region. The diameter of the lower region maybe 0.25 inch or less, preferably 0.1 inch or less, more preferably 0.1to 0.08 inch, and most preferably 0.08 to 0.039 inch. Smaller diametermicrobanding regions are feasible and are within the province of thisdisclosure. The length of the lower region is typically between 5% to25% of the length of the tube. In one aspect, the tube may also includea seal with a central opening which can be plugged and unplugged.

[0020] In accordance with the present invention, a bucket is providedfor holding an ultracentriflige tube. The bucket comprises upper andlower regions which may be of successively smaller diameters, may haveinserts to successively decrease the inner diameter, or may be ofuniform internal diameter, and may further comprise a third region whichattaches the bucket to a rotor.

[0021] Further in accordance with the present invention, a method isprovided for concentrating microorganisms. As used herein, the term“microorganisms” is intended to include viruses, myoplasmas, rickettsia,yeast and bacteria. The method comprises ultracentrifugation of a samplecontaining the microorganism in an ultracentrifuge tube describedherein. The ultracentrifugation may include the formation of densitygradients and/or the staining of the microorganism(s). In one aspect,the staining can be used to distinguish the DNA or RNA content of avirus. The banding of the microorganisms upon ultracentritugation can beused to identify the microorganisms.

[0022] In a further aspect of the invention, the concentratedmicroorganisms are further characterized by conventional techniques suchas mass spectrometry, flow cytometry, optical mapping, isopycnic bandingdensities, fluorescence, restriction enzyme analysis, genome size,enzymatic or chemical resistance/susceptibility, immunochemistry and thelike. In another aspect of the invention, the amount or titre of themicroorganisms can be determined.

[0023] In accordance with the present invention, a system is providedfor measuring fluorescence from a sample in a centrifuge tube. In oneembodiment, the system includes a centrifuge tube, a light source, suchas a laser, and a detector to detect light passing through the sample oremitted from the sample upon light passing through it. Optical filtersselect and separate the exciting and emitted wavelengths of light.

[0024] The internal surfaces of the tubes and especially the funnelportion must be very smooth in order to prevent small virus particlesfrom being retarded by surface irregularities, and in addition, thesurfaces must be treated so that infectious particles are not adsorbed.Polishing of plastic surfaces is done by brief exposure to a solventvapor. For example, polycarbonate is polished by brief exposure toheated methylene chloride gas. Plastic surfaces are modified to preventadsorption of infectious particles by exposure to dilute solutions ofproteins such as bovine serum albumin or gelatin, or to charged polymerssuch as heparin or derivatives of heparin. Both of these procedures arewell known to those practiced in the arts.

[0025] Further in accordance with the present invention, a system isprovided for counting particles concentrated in a small volume. Thesystem includes a container in which the particles are concentrated, acapillary tube, two pumps, means for moving the container relative tothe capillary tube, a flow cell, a light source and detector.Alternatively, fluid may be moved by gas pressure instead of pumps.

BRIEF DESCRIPTION OF THE DRAWINGS

[0026]FIG. 1 shows an S-rho plot for a typical tissue and forrepresentative viruses.

[0027] FIGS. 2A-2C are a diagrammatic representation of one embodimentof a centrifugal microbanding tube and its use.

[0028] FIGS. 3A-3G show alternative embodiments of microbanding tubesand use in a rotor (3F).

[0029]FIGS. 4A and 4B illustrate a centrifuge swinging bucket designthat allows higher speed fractionation of large sample volumes.

[0030]FIG. 5 illustrates a complete system including verticalmonochromatric laser illumination, goniometer and X-Y stage forsupporting and positioning microbanding tube, microbanding tube withbanded viruses particles, and camera system.

[0031]FIG. 6 illustrates a complete system including interference filterlight sources, light pipe illumination, digital data acquisition, andCRT data presentation.

[0032]FIG. 7 illustrates a method for recovering banded virus particlesusing a micropipette, and counting them by flow cytometry.

[0033] FIGS. 8A-8C illustrate details of band recovery.

[0034] FIGS. 9A-9F illustrate one embodiment of a closure for swingingbucket rotor centrifuge tubes and detection of a sample with respect tothe tubes.

DETAILED DESCRIPTION OF THE INVENTION

[0035] The invention is directed to methods of identifying and measuringthe presence of microbial agents such as bacteria and viruses inbiological samples. The methods include centrifugation steps to purifythe microbial agents in a very small volume. The agents are then assayedby means such as isopycnic banding density, fluorescence or massspectrometry.

[0036] It is an object of this invention to develop integrated systemsand methods in which suspensions containing microorganisms, includinginfectious agents, are stained with one or more fluorescent dyes, inwhich a step or continuous gradient is automatically formed duringcentrifugation, in which the microorganisms are centrifuged away fromthe stain-containing suspending medium and are washed free of externalstain, are concentrated in a gradient of very small cross section,separated according to their isopycnic banding densities, their bandingdensities determined, and the microorganisms detected by fluorescence.

[0037] It is a further object of this invention to concentratemicroorganisms, including infectious agents, into microbands by a factorof 1-5,000.

[0038] It is a further object of this invention to expose themicroorganisms, such as infectious agents, to reagents includingdetergents, surfactants, enzymes, or organic solvents contained indistinct zones in a density gradient to dissolve or disassemblecontaminating particles to prevent them from banding with themicroorganisms, and to separate stained particles from the free stain ofthe initial sample volume.

[0039] It is a further object of this invention to use one or more dyeswhich bind differentially to RNA, single stranded DNA or double strandedDNA to allow these to be distinguished by their fluorescent spectra.

[0040] It is a further object of this invention to provide for theconcentration of banded microorganisms, for example infectious agents,by resuspending the banding gradient, which is typically 0.04 mL, toapproximately 4 mL in water or a very dilute buffer, and pelleting themicroorganisms one or more times to provide a concentrated pellet freeof gradient materials for mass spectrometric analysis, for counting byepifluorescent microscopy or by flow cytometry.

[0041] It is a further object of this invention to provide means for thediagnosis of infectious diseases which minimize exposure of laboratorypersonnel to infectious agents.

[0042] It is an additional object of this invention to provide means forpreparing nucleic acids from small quantities of microorganisms,including infectious agents, to determine the masses of intact nucleicacid molecules, and for characterization of fragments produced byrestriction enzymes using either flow cytometry or epifluorescencemicroscopy.

[0043] It is an additional object of this invention to determine thebanding densities of the microorganisms, such as infectious agents,accurately by reference to the positions of calibrated particles addedto the gradients.

[0044] For ease of description, the invention will be described withreference to viruses as the microorganisms. It will be understood thatthe invention is also applicable to other microorganisms, includingmycoplasmas, yeast and bacteria. The invention is particularly suitedfor the identification of infectious agents, and will be described inthis context.

[0045]FIG. 1 is a graph depicting the sedimentation coefficients andisopycnic banding densities of subcellular organelles and viruses toillustrate the concept of the “Virus Window” (Anderson, 1966). It isevident that viruses have a relatively narrow range of sedimentationcoefficients and banding densities and may be isolated from a tissuehomogenate or from blood in a high state of purity using high resolutionS-ρ separation systems. For a complete description of high resolutionS-ρ centrifugal methods and of centrifuge development for virusisolation, refer to National Cancer Institute Monograph 21, TheDevelopment of Zonal Centrifuges and Ancillary Systems for TissueFractionation and Analysis, U.S. Department of Health, Education andWelfare, Public Health Service, 1966. This work describes S-ρ separationtheory and systems, and the use of colored plastic beads of gradeddensities as density markers.

[0046] In practice, a blood sample or tissue homogenate is centrifugedto sediment all particles having sedimentation rates higher than that ofthe particle or particles to be analyzed. For viruses, that meansparticles of circa 10,000 S and above are discarded. The supernatantafter such a separation is then used as the sample for second dimensionisopycnic banding separations carried out in microbanding tubes asdescribed here, using centrifugation conditions which will sediment andisopycnically band all known infectious particles.

[0047] One picomole of virus would contain 6.022×10¹¹ viral particles,while 6×10⁹ virions would contain 1 picomole of a viral coat proteinpresent in 100 copies per virion. Quantitative polymerase chain reaction(PCR) has been used to demonstrate that in many infectious diseases>10⁸virus particles are present per mL of plasma or serum. Hence, if thevirions from a 5-10 mL biological sample containing 10⁸ virions/mL areconcentrated to a microliter or two, and then applied to a very smalltarget area, individual viral proteins can be detected usingMALDI-TOF-MS Krishmanurthy et al., 1996; Holland et al., 1966). Usingelectrospray techniques, samples containing 10⁶ virus particles/mL canbe detected, while with flow cytometry and immobilized DNAepifluorescence microscopy, even fewer particles are required (Hara etal., 1991; Henres and Suttle 1995). The application of these methods tobacteria may require a preseparation of proteins to reduce thecomplexity of the sample. In mass spectrometry, detection has been bycharged ion detection, and the limitations of such detection have setthe upper limits to the size of proteins and nucleic acids that can bedetected. Mass spectrometric methods have now been described which allowmasses of biological particles above 100,000 daltons to be measured.

[0048] In order to work with such levels of virus, the virus must beconcentrated into a very small volume. This concentration isaccomplished during the second dimension of centrifugation (theisopycnic banding step) by banding the virus using a centrifuge tubespecially designed to concentrate the virus into a microband afterpassage through gradient layers that wash the particles and expose themto selected reagents. An example of such a microbanding centrifuge tubeis shown in FIGS. 2A-2C. FIG. 2A illustrates diagrammatically a hollowtransparent centrifuge tube 1 with an upper sample volume 2, gradinginto a serrated funnel region 3 having successively tapered andparallel-wall sections 4-6, constricting down to a narrow tubularmicrobanding region 7. The serrated funnel region 3 is an improvementover centrifuge tubes which simply taper from top to bottom withoutincluding a serrated region. By serrations is meant, for example,concentric rings or edges or lips. These rings, edges or lips arepreferably continuous around the inner diameter of the centrifuge tube,but this is not required. For example, three projections from the innerwall of the centrifuge tube spaced equally around the diameter could beused to hold a disk in place. The term serrations is meant to includesuch possibilities but does not include a straight tapering with norings, edges, lips or projections on the inner surface of the centrifugetube. The serrations can be used as rests onto which disks can be placedto separate two or more layers of liquid. Although disks can be placedinto tubes which simply taper without serrations, the disks in suchtapered tubes can be easily tipped up on one edge by pushing down on theopposing edge. This would cause a premature mixing of the layers whichare to be separated by the disks. The serrated region allows disks tolie flat and prevents the disks from being accidentally tipped up. As anexample, the centrifuge tube may be 3.45 inches from top to bottom, havean outer diameter at the top of 0.562 inch, and have an inner diameterin the bottom microbanding region 7 of 0.064 inch. Such a tube issuitable for use in an SW41 Ti (Beckman) rotor. The inside surface ofthe tube is preferably polished using conventional techniques, includingvapor polishing, so that the virus particles do not stick to the wall ofthe tube. Additionally, the internal surfaces of the tubes may be coatedwith a protein or polymer to prevent particle adhesion, as is well knownin the art.

[0049]FIG. 2B illustrates how the tube is loaded at rest with a seriesof fluids of decreasing physical density. The tube shown comprises aseries of serrations onto which can be laid disks to separate one layerof fluid from the next layer of fluid. Liquid 8 is denser than anyparticle to be recovered, and is used to partially fill the microbandingregion 7. When a less dense fluid 9 is pipetted in with a micropipet anair bubble 10 (wherein by air is meant atmospheric air or another gas)may be left to keep the fluids 8 and 9 separate. Similarly when thefirst overlay fluid 11 is introduced, air bubble 12 may be left inplace, thus keeping the three liquids separate until centrifugation iscommenced. A tube with an inner diameter of 0.064 inches in microbandingregion 7 is suitable for allowing an air bubble to be left in place toseparate two layers of liquid. Alternatively, the air bubbles may beleft out and the fluids allowed to diffuse together to create a densitygradient. Fluid 11 is covered with a light porous plastic disk 13,preferably of sintered polyethylene or polypropylene, which fits inplace in the first serration. A fluid 14, less dense than fluid 11,which may contain one or more reagents, is then introduced, and coveredwith disk 15, followed by even less dense liquid 16 which is coveredwith disk 17. The entire system is stable until centrifuged. Before usethe sample layer 18, which has a density less than that of fluid 16 isthen added up to level 19. The tubes are then centrifuged at high speedin metal shields, typically with water or other liquid added to theshields. In addition, the tubes may be supported by fitting adapterswhich fill the space between the tubes and the shields, and water may beadded to fill any spaces between the tubes, adapters and shields toprovide additional support. Optionally the tubes may be capped (as shownin FIGS. 9A-9F, described in further detail below), to minimize thechances of operator infection.

[0050]FIG. 2C illustrates diagrammatically a tube after centrifugation.The porous separation disks 13, 15, and 17 have risen to the top of thetube, and sample layer 18 is cleared of virus, and the original stepgradient has changed, by diffusion, into one of a series of shallowgradients. In addition, gas bubbles 10 and 12 have also movedcentripetally, and fluids 8 and 9 have come into contact to form a steepgradient by diffusion. As centrifugation proceeds, the slope of thisgradient diminishes, producing a banding gradient of a width suitablefor banding the infectious agents. For cesium chloride gradients, thedensities typically range from 1.18 to 1.55 g/ml. These gradient stepsmay not only contain reagents to dissolve non-viral particles, but alsoserve to wash excess fluorescent dye away from the particles. Forexample, various detergents or enzymes such as proteases may be addedeither to the sample layer 18 or to other layers such as 14 or 16.Fluorescent dyes may also be present in these regions. The free dye willnot enter the lower, more dense regions in which the virus bands andtherefore the centrifugation will purify the viruses from all of thereagents which may be present in the upper, less dense layers. Aftercentrifugation, the microbanding region of the tube contains the upperportion of the banding gradient 27, banded virus 28 (including any dyebound to the virus or viral nucleic acid) and lower dense portion of thebanding gradient 29, and the gradient formed between them by diffusion.

[0051] FIGS. 3A-3G illustrate alternative embodiments of tubes usefulfor microbanding of viruses and bacteria and all have a serratedinternal construction which allows one or more light barriers to bepositioned and retained at rest. Tubes shown in FIGS. 3A-3D and 3G aredesigned to be centrifuged in swinging bucket rotors so that the tubesare horizontal during centrifugation and vertical at rest. The tubeshown in FIG. 3A is the more conventional design with a sample reservoir31, a serrated finnel region 32, and a microbanding section 33. The tubeshown in FIG. 3B is similar to that of FIG. 3A, but it is supported in acentrifuge shield by a support insert 34 which may be of plastic ormetal. The tubes shown in FIGS. 3B-3E fill a rotor chamber completely.The tube of FIG. 3C has an opaque bottom section 35 which absorbsscattered light, while that shown in FIG. 3D has a bulbous section 36 atthe bottom of the microbanding tube 37 to contain an excess volume ofthe fluid forming the dense end of the gradient, thus stabilizing thegradient. The tube shown in FIG. 3E is designed to be centrifuged in anangle head rotor as shown in FIG. 3F, and has a linearly continuous wall38 along one side positioned in the rotor so that particles may readilyslide down to microbanding region 40. The tube shown in FIG. 3Gillustrates how a very large microbanding tube may be fabricated.

[0052] FIGS. 4A-4B illustrate how the tube of FIG. 3G may be centrifugedat higher speed than tubes having a constant radius from top to curvedbottom. This is accomplished by using a metal, plastic or carbon fibershield 45 which matches the dimensions of tube 46. The shield has a cap47 and the shield or bucket swings on integral attachment 48, as isconventionally done in high speed swinging bucket rotors. Tip 49 of theshield is much smaller diameter than the upper section of the shield,has much less mass swinging at its maximum radius, and hence can reachmuch higher speeds than is the case with shields of uniform internaldiameter. This makes possible isolation of trace amounts of virus frommuch larger volumes than would otherwise be the case. Duringcentrifugation using rotor 50 driven by drive 51, shield and tube 52assume a horizontal position as shown.

[0053] The microbanded viruses can be analyzed at this stage or they canbe collected, diluted, and further processed. To analyze the microbandedviruses at this stage, they can be detected by a system as shown in FIG.5. For example, the isopycnic banding step or an earlier step may haveincluded a fluorescent dye or fluorescent dyes within the solution withwhich the virus was mixed or through which the virus was centrifuged.Dyes are known with which intact viruses may be stained and which candistinguish between RNA, DNA, single stranded nucleic acid and doublestranded nucleic acid thereby allowing one to detect the presence orabsence of an infectious agent, and further to determine which type ofvirus one has purified. The apparatus of FIG. 5 can be used to analyzethese stained particles.

[0054] A scanning and detection system is illustrated schematically inFIG. 5 where microbanding tube 60 is held in a vertical position onmount 61 supported by goniometers 62 and 63 which are in turn supportedby X-Y movements 64 and 65 in such a manner as to align and center themicrobanding section of tube 60 with respect to laser beam 66. Laserbeam 66 is generated by a laser 67, which may be an argon ion laserproducing coherent light at 458, 488, 496, 502, and 515 nm. The beampasses through an interference or other filter 68 to isolate onewavelength, and is reflected down into the microbanding tube by dichroicmirror 69. The fluorescent banded particle zone 70 is photographed orelectronically scanned by camera 71 through emission filter 72. Theentire system may be enclosed to eliminate stray light, and filters 68and 72 may be replaced by filter wheels (not shown) to optimizedetection using fluorescent dyes which absorb and emit at differentwavelengths, or to distinguish ssDNA, dsDNA and RNA by differences inthe spectra of emitted fluorescent light. Electronic shutters may beattached to the laser to minimize sample exposure to light and to thecamera to control exposure. The goniometers and X-Y movements may alsobe motor driven and remotely controlled, and the entire system may becontrolled by a computer (not shown).

[0055]FIG. 6 illustrates a different version of the scanning systemwhich can cover all of the visible spectrum and on into the nearultraviolet. Microbanding tube 80 is aligned in a fixed support betweentransparent intensity equilibrators 81 and 82 attached to light pipes 83and 84 which are in turn attached to intensity equilibrator 85illuminated through filter 86 by condensing lens 87 and light source 88.Filter 86 is one of a set attached to filter wheel 89 indexed by motor90. The result is uniform illumination from two sides of one or morebands 91, 92 and 93. The image is captured through emission filter 94 bydigital camera 95 and the image stored, processed and displayed bycomputer 96 on CRT 97. Filter 94 may be replaced by a filter wheelidentical to 89 and 90 so that, with both an excitation filter wheel andan emission filter wheel and a wide spectrum light source such as axenon lamp or a halogen lamp, a wide variety of combination of excitingand emitting light may be chosen, which in turn makes possible use of awide variety of fluorescent dyes. Both fluorescent light and lightscatter at a chosen wavelength may be employed for particle detection.This arrangement facilitates distinction between ssDNA, dsDNA and RNA.

[0056] The processed image 98 may be displayed to show a picture of thetube and contained bands 99, 100 and 101. The amount of light from eachband may be integrated and displayed as peaks 102, 103, and 104, and inaddition the integrated values may be displayed digitally (not shown).The entire system including shutters on the light source and camera (notshown), filter movement and positioning, and focusing of the camera maybe digitally controlled by computer 96.

[0057] Display bands 99 and 101, representing centrifuge tube bands 91and 93 may be fluorescent or non-fluorescent density marker beads ofknown density, and the virus band 92 represented by display band 103.The banding density of the virus may be determined by interpolation fromthe positions of the density markers. When non-fluorescent densitymarkers are used, these are detected by scattered light using identicalfilters at positions 86 and 94. A second image using suitable anddifferent filters is then captured which is comprised solely offluorescent light. The two images are electronically inter-compared andthe physical density of the infectious agent determined byinterpolation.

[0058] At this stage, the virus can be identified as being a DNA virusor an RNA virus, and if a DNA virus it can be determined whether it issingle stranded or double stranded. Furthermore, the density of thevirus can be determined. This data can be used to help identify the typeof virus which has been purified. Nevertheless, it may be desirable ornecessary to gather more data to fully determine what the exact virus isand also to determine the original viral titre.

[0059]FIG. 7 illustrates diagrammatically counting of individualfluorescent particles recovered from a tube 110 containing zones ofbanded virus 111 and 112 after all fluid above the banding gradient hasbeen removed and replaced. The tube is placed in a tube holder 113, andan overlay of deionized water or very dilute buffer 114 is introducedabove the gradient supplied through tube 115 to replace the volume drawnup in the probe 117. The tube may be closed at the top by a plasticclosure 116. The capillary probe 117 is held stationary, and themicrobanding tube 110 is slowly raised under it. The tube holder 113 ispart of a precision drive mechanism 118 and associated stepping motor119 that moves the tube holder vertically at a very slow andcontrollable rate. A slow steady stream of fluid is drawn intoconstriction 120 which is centered in sheath stream 121 provided by pump122. The result is a constant flow of fluid through flow cell 123 with afine virus containing stream in the center, elongated and extended bythe flowing sheath. A second pump 124 withdraws fluid upward at aconstant rate from the flow cell, which rate is greater than the rate atwhich piston pump 122 injects fluid into sheath 121. The difference inthe rates of pumps 124 and 122 is made up by the fluid coming throughcapillary probe 117. The fluid coming through capillary probe 117 is amixture of virus plus fluid from the overlay which is introduced viatube 115.

[0060] The flow cell 123 is illuminated by laser beam 125 produced bylaser 126, that passes through exciting filter 127. Emitted light isisolated by emission filter 128 and detected by a photomultiplier 129.The output from the photomultiplier 129 is integrated at intervals bycomputer 130, and the integrated signal vs. time is displayed on CRT131. When two viral bands are present, two peaks such as 132 and 133,are displayed. Depending on the number of fluorescent particles present,the signal generated from a band may be integrated into a peak, or, ifthe suspension is sufficiently dilute, the particles may be countedindividually, the values binned, and the integrated results displayed.

[0061] In order to count the particles as just described, it isnecessary that the virus particles are greatly diluted as they passthrough flow cell 123. FIG. 8 illustrates diagrammatically how theproblem of making an initial dilution of a very small-volume virus bandfor counting individual particles is accomplished. FIG. 8A shows a tube110 as in FIG. 7, with a section indicated which is shown enlarged inFIG. 8B, which in turn shows the section of that panel enlarged in FIG.8C. As the movement upward of the microbanding tube causes the capillarytube to move toward the tube bottom, the difference in pumping rates ofthe two pistons attached to the flow cell causes fluid to flow up thecapillary where it is diluted as described by the combined action ofpumps 122 and 124 of FIG. 7. However, the amount of fluid drawn into thecapillary 117 is much greater than the volume of fluid effectivelydisplaced from the banding gradient by the relative movements of thecapillary and the microbanding tube. This volume is replaced by fluidflowing into tube 115 though cap 116 which is initially allowed to flowin until the tube 110 is full. This fluid is much less dense than thedensity of the fluid at the top of the gradient in the microbandingregion, and causes minimal disturbance in the gradient. As shown in FIG.8B, the capillary 117 slowly approaches virus band 144, and, as shown inFIG. 8C, a small amount of gradient liquid 145 is diluted by a largeramount of supernatant fluid 114 as it flows up the capillary. In thismanner, a sharp band of virus particles 144 is diluted and moves throughthe flow cell as, volumetrically, a larger band, but with littleeffective loss of resolution. This technique provides the dilutionnecessary to make counting of individual virus particles feasible andaccurate. The amount of dilution can be controlled such that theconcentration of microorganisms in the capillary tube is less thanone-half or one-tenth, or one-hundredth, or one-thousandth, or oneten-thousandth, or one-millionth, or one-billionth of the concentrationin the band in the lower region of said tube.

[0062] In addition to counting the particles or determining the titre ofa virus, the amount of DNA in the virus or other microbe can bedetermined for individual particles. In this aspect of the invention,the amount of DNA in the particles is measured by flow fluorescenceanalysis (Goodwin et al., 1993) or epifluorescence analysis (Jing etal., 1998). In this manner, yeast, bacteria, mycoplasm and virus can bedistinguished as groups, For example, it is known that viruses contain5-200×10³ bases or base pairs, E. coli, a typical bacterium, contains4×10⁶ base pairs, while a typical yeast cell contains 1.3×10⁷ basepairs. Thus, an estimate of the amount of DNA or RNA present allows theclass of an infectious agent to be determined.

[0063] Thus, the size of a genome can be determined. In this embodimentof the invention, the genome is extracted from the microorganism bandand immobilized on a solid support. The immobilized DNA is stained andelectronically imaged using an epifluorescence microscope (Jing et al.,1998). The length of the individual nucleic acid molecules can then bemeasured.

[0064] The technique of microbanding is useful not merely for stainingthe virus with dyes and being able to count the virus particles. Oncethe viruses from a biological sample have been highly purified andconcentrated by the two dimensional centrifugation technique asdescribed above using microbanding centrifuge tubes, the viruses areamenable for use in many other assays.

[0065] When an infectious agent is banded in a microbanding tube, theband may also be judiciously removed using a capillary needle in avolume of a few microliters, diluted to 5 mL or more with very dilutebuffer or deionized water to dilute the gradient materials by a factoras large as 1,000, and then pelleted in a fresh microbanding tube. Thesupernatant may then be carefully withdrawn by a suction capillary, andthe virus or other agent resuspended in approximately 1 microliter usinga syringe made, for example, of fine Teflon® tubing fitted with a verysmall stainless steel wire plunger to fit. The sample may then betransferred to a mass spectrometer target, mixed with a matrix dye, andused for matrix assisted laser desorption ionization time of flight massspectrometry (MALDI-TOF-MS) to determine directly the masses of viralcoat proteins or of bacterial cell proteins. Technology described forsample concentration may also be applied, without a matrix dye, toelectrospray or other mass spectrometric analysis systems, including thedetection of intact viral mass.

[0066] A system similar to that shown in FIG. 7 may also be used toproduce the equivalent of molecular restriction fragment length maps ofDNA molecules using restriction enzymes. For this work, virus orbacterial particle bands may be diluted and sedimented as described,after which the DNA may be extracted using detergents or other reagentswell known in the art, treated with a restriction enzyme and afluorescent dye, and the fragment sizes determined by flow cytometry(Goodwin et al., 1993; Hammond et al., U.S. Pat. No. 5,558,998).Extracted DNA may also be immobilized on a solid support, stained with afluorescent dye, and photographed using an epifluorescence microscope todetermine the length of DNA molecules. The preparation may then betreated with a restriction endonuclease, and the number and lengths ofthe oligonucleotide fragments determined (Jing et al., 1998). These dataare then compared with a database listing the expected fragment lengthsfor different viral or bacterial species to identify each agent. DNAfragment lengths may also be determined by gel electrophoresis.

[0067] Fluorescence labeled antibodies may also be added to the particlesuspension studied, and the presence or absence of the label inisopycnically banded particles determined. This approach is useful forspecific identifications, and the use of a set of antibodies labeledwith dyes having different and unique spectral characteristics allowsthe presence or absence of a series of agents to be determined.Alternatively antibodies labeled with chelators for rare earth's such asEuropium and Terbium may be employed, in which case delayed fluorescenceis measured.

[0068] Serum or plasma typically has a physical density between 1.026and 1.031. Viruses typically have banding densities between 1.17 and1.55 in cesium chloride, and at much lower densities in iodinatedgradient materials such as lodixanol or sucrose (Graham et al., 1994).The intermediate wash and reagent layers between the sample and thebanding gradient must therefore have densities less than the density ofthe lightest virus to be banded. Buffers used to dissolve gradientmaterial for virus isolation include 0.05 M sodium borate, and 0.02 Msodium cyanide, both of which prevent bacterial growth.

[0069] With human serum or plasma, centrifngation sufficient to removeplatelets and other particles having sedimentation coefficients ofapproximately 10⁴ S is used before banding of virus particles.

[0070] The banding density of virus particles depends on the nucleicacid/protein ratio, and the presence or absence of lipids andlipoproteins. Hence attachment of specific identifying antibodieslabeled with fluorescent dyes should not only allow identification byfluorescence but by a banding density change.

[0071] To assist in identifying particles by density, fluorescentparticles of known density may be included in the sample as shown inFIG. 6. These particles may include known fixed and fluorescentlystained virus or bacterial particles of known banding density, or verysmall fluorescently labeled or non-fluorescently labeled plastic beads.When polystyrene latex particles are coated with antibodies, theirbanding densities are increased appreciably, and the density may befurther increased by reaction of the antibody-coated particles with theantigen for which the antibodies are specific (Anderson and Breillatt,1971). Antibody-coated fluorescent polystyrene beads may therefore beused not only to locate virus particles but to identify them.

[0072] The stains which are currently most useful are described in theHandbook of Fluorescent Probes and Research Chemicals, R. P. Haugland,ed., Molecular Probes, Inc., Eugene, Oreg. (1996), which lists theabbreviated dye names, their chemical names, absorption and emissionmaxima, and the filter combinations most used.

[0073] For work with human pathogens, safe operation and containment areimportant (Cho et al., 1966). Use of swinging bucket rotors, whileoptimal from a physico-chemical point of view, require extensivemanipulation and have more parts than an angle-head rotor. The tubesdescribed in FIG. 3E are designed to be used in angle-head rotors, andallow sedimenting particles to travel along a wall at one unchangingangle. Such rotors are easier to use and handle in containment than areswinging bucket rotors, however, sedimentation in an angle head rotor isfar from ideal. Hence, the development of methods and devices for safelyworking with swinging bucket rotors is important.

[0074] High speed centrifuge tubes are notoriously difficult to sealeffectively and are a potential source of infection to laboratorypersonnel. In practice, nearly all high speed swinging bucket rotortubes are not themselves sealed, but are enclosed in a metal bucketwhich is sealed with a metal cap which does not seal the tube. Thecentrifuge tubes are therefore open when loaded, moved to the centrifugerotor, inserted, and removed. It is very difficult to decontaminate theoutside of an open tube containing a density gradient without disturbingthe gradient. In the present application it is desirable to be able toeffectively seal the plastic tubes is such a manner that the outsidesurfaces can be cleaned with a suitable disinfectant before the tubesare inserted into centrifuge shields, and to be able to handle themsafely until they are scanned.

[0075] Sealing is done, as shown in FIG. 9A, by inserting an annularring seal 151, having a physical density less than that of water, intotube 152. Ring 151 is slightly tapered so that it fits very tightly intotube 152, and has a center hole which can be plugged and unplugged. Inone embodiment, the center hole is threaded to accept a short, plasticflat-head screw. Initially two gradient components, including a lightersolution 153 and a denser solution 155, are introduced to the bottommicrobanding region with a small air bubble 154 between, as previouslydescribed. As shown in FIG. 9B, the solution 156 containing theinfectious agent or other particles is then introduced through tube 157,leaving bubble 158 to separate the sample from the upper gradientsolution. The volume of sample introduced does not completely fill thecentrifuge tube, leaving space 159 empty. The tube is then sealed, asshown in FIG. 9C with, for example, a plastic flat head screw 160,leaving air bubble 160 in place. The outside of the tube is thensterilized by immersing it in a disinfectant such as a sodiumhypochlorite or hydrogen peroxide solution, followed by a water wash andgentle drying—all with the tube in an upright position. Aftercentrifugation, as shown in FIG. 9D, the plastic upper seal has beendriven downward by centrifugal force a small distance as the entrappedair rises around the plastic seal. However, since the seal has a densityless than water, it is retained at the top of the liquid sample, leavinga small lip 160 which may be grasped with a hemostat to remove the tubefrom the centrifuge shield. During centrifugation, the infectiousparticles are sedimented out of liquid 163 and produce band 164 in thegradient. The screw in the ring seal is then removed, and, as shown inFIG. 9E, the supernatant liquid 165, which may contain a fluorescentdye, is removed through tube 166, leaving meniscus 167. As shown in FIG.9F, a laser beam 168, entering the tube from above, is then aligned withthe tube, causing the banded infectious agent to emit fluorescent lightfor detection as previously described.

[0076] When a step gradient containing various reagents in addition tothose used for isopycnic banding is employed, as illustrated in FIG. 2,the discs used to separate the several solutions rise to the top andwould not allow the use of vertical laser illumination as shown in FIG.9F without removing the seal and the discs. In this instance, sideillumination, as illustrated in FIG. 6 would be employed.

[0077] The laser or delayed fluorescence systems can be completelycontained, the mechanical operations done remotely through smallstepping motors, and the tubes moved in and out of the contained systemunder remote control.

[0078] These techniques can be combined with mass spectrometry andfluorescence-based restriction fragment mapping to allow rapid diagnosisand identification of infectious agents. However, the estimates of themasses of individual proteins are generally taken from publishedsequence data, and do not include numerous posttranslationalmodifications. Mass spectrometric data bases must be created to includeactual mass measurements of different microorganisms. In addition,virion protein mass measurements will allow the detection of manygenetic variants. However, for many studies of microorganisms, includingdevelopment of data bases, the key problem has been the development ofmethods for systematically providing highly concentrated and purifiedmicrosamples of microorganisms from patient samples, natural waters, andfrom tissue culture fluids. This problem is solved by the presentinvention.

[0079] The present invention is described by reference to the followingExample, which is offered by way of illustration and are not intended tolimit the invention in any manner. Standard techniques well known in theart or the techniques specifically described below were utilized.

EXAMPLE

[0080] To illustrate the use of microbanding tubes, experimental studieshave been carried out using a small single-stranded non-pathogenic DNAvirus φX 174. Approximately 10¹⁰ virus particles which had been purifiedby isopycnic banding in CsCl in a microbanding tube such as shown inFIG. 2A were suspended in 5 mL of 0.05 M borate buffer, were pelleted ina microbanding tube at 35,000 rpm in a swinging bucket rotor, and wereresuspended in approximately 3 μL for analysis. The analyses were doneon a PerSeptive Biosystems DESTR instrument with an extraction delaytime set at 150 nseconds using a matrix of sinapinic acid. Bovineinsulin (Mw=5,734.59) and horse heart apomyoglobin (Mw=16,952) were usedas 1 and 2 pmole standards. The results are shown in Table 1. The φX 174masses for virion capsid proteins F, G, H and J are calculated frompublished sequence data. The differences between the calculated andexperimental values for F and H are probably due to posttranslationalmodifications. The probability that an unrelated virus could havesubunits of the same masses listed is vanishingly small. However, evenmore definitive protein identifications can be made by treating viralproteins with proteolytic enzymes such as trypsin and determining themasses of the peptide fragments produced. Computer programs areavailable which calculate the sizes of fragments of proteins of knownsequence by well characterized enzymes. Such programs include ProteinProspector (available from the University of California, San Francisco)and ProFound (available from Rockefeller University). TABLE 1 MassSpectrometric Analysis of φX 174 Virion Proteins Calculated ExperimentalProtein Mass Mass Mass Difference % Difference F 48,351.53 48,407.4+55.9 0.12% G 19,046.73 19,046.7 0.0 0.00% H 34,419.25 34,466.1 +46.90.14% J 4,095.78 4,097.03 +1.2 0.03%

[0081] This example demonstrates that highly purified and concentratedsuspensions of microorganisms can be isolated from biological samplessuch as, but not limited to, patient samples such as plasma, urine,feces and tissues, natural water and tissue culture fluids. This examplefurther demonstrates that such purified and concentrated microorganismscan then be identified, for example, using mass spectrometry to identifyviruses.

[0082] While the invention has been disclosed in this patent applicationby reference to the details of preferred embodiments of the invention,it is to be understood that the disclosure is intended in anillustrative rather than in a limiting sense, as it is contemplated thatmodifications will readily occur to those skilled in the art, within thespirit of the invention and the scope of the appended claims.

LIST OF REFERENCES

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What is claimed is:
 1. A method for distinguishing betweensingle-stranded DNA viruses, double-stranded DNA viruses and RNA virusespresent in a biological sample containing viruses, which comprisescontacting dyes which can distinguish between said viruses with virusesin said sample, concentrating said viruses by i) adding a samplecontaining said viruses to an ultracentrituge tube and ii) centrifugingsaid sample in said tube to concentrate said viruses, saidultracentrifuge tube comprising an upper region, a middle region and alower region, wherein an inner diameter of said upper region is largerthan an inner diameter of said lower region, wherein said upper regionis separated from said lower region by said middle region having adecreasing diameter from said upper region toward said lower region andwherein said lower region has a closed bottom, and detecting the bounddyes in a band of virus, whereby the type of nucleic acid present in theviruses is determined.
 2. The ultracentrifuge tube of claim 1, whereinsaid middle region comprises one or more serrations.
 3. Theultracentifuge tube of claim 1, wherein said lower region has an innerdiameter small enough to trap an air bubble between two layers of liquidsuch that the air bubble will keep said two layers of liquid separate solong as said centrifuge tube is at rest.
 4. The method of claim 1,wherein said dyes are added to said sample.
 5. The method of claim 1,wherein such dyes are added to a layer of fluid added to the tube beforethe addition of said sample and said viruses contact said dyes duringcentrifugation.
 6. The method of claim 1, wherein said dyes arefluorescent and said bound fluorescent dyes are detected by passing anexciting fluorescent light through said band of virus and determining awavelength of peak intensity of emitted fluorescent light from said bandof virus.
 7. The method of claim 6, which further comprises removingunbound dyes from said tube prior to determining said wavelength of peakintensity.
 8. The method of claim 1, wherein said bound dyes aredetected by passing an exciting light through said band of virus anddetermining the spectral distribution of the emitted light.
 9. A methodfor determining an infectious agent titre in a biological sample, whichcomprises measuring the intensity of emitted fluorescent light of claim6.
 10. A method for distinguishing between single-stranded DNA viruses,double-stranded DNA viruses and RNA viruses present in a biologicalsample containing viruses, which comprises contacting dyes which candistinguish between said viruses with viruses in said sample,concentrating said viruses by i) adding a sample containing said virusesto an ultracentrifuge tube and ii) centrifuging said sample in said tubeto concentrate said viruses, said ultracentrifuge tube comprising anupper region, a middle region and a lower region wherein an innerdiameter of said upper region is larger than an inner diameter of saidmiddle region, wherein (i) an inner diameter of said middle region islarger than an inner diameter of said lower region or (ii) the innerdiameter of said middle region is the same as the inner diameter of saidlower region, wherein that inner diameter is small enough to trap an airbubble between two layers of aqueous liquid such that the air bubblewill keep said two layers of aqueous liquid separate so long as saidcentrifuge tube is at rest, and wherein said lower region has a closedbottom, and detecting the bound dyes in a band of virus, whereby thetype of nucleic acid present in the viruses is determined.
 11. Themethod of claim 10, wherein said dyes are added to said sample.
 12. Themethod of claim 10, wherein such dyes are added to a layer of fluidadded to the tube before the addition of said sample and said virusescontact said dyes during centrifuigation.
 13. The method of claim 10,wherein said dyes are fluorescent and said bound fluorescent dyes aredetected by passing an exciting fluorescent light through said band ofvirus and determining a wavelength of peak intensity of emittedfluorescent light from said band of virus.
 14. The method of claim 13,which further comprises removing unbound dyes from said tube prior todetermining said wavelength of peak intensity.
 15. The method of claim10, wherein said bound dyes are detected by passing an exciting lightthrough said band of virus and determining the spectral distribution ofthe emitted light.
 16. A method for determining an infectious agenttitre in a biological sample, which comprises measuring the intensity ofemitted fluorescent light of claim
 13. 17. A method for distinguishingbetween single-stranded DNA viruses, double-stranded DNA viruses and RNAviruses present in a biological sample containing viruses, whichcomprises contacting dyes which can distinguish between said viruseswith viruses in said sample, concentrating said viruses by i) adding asample containing said viruses to an ultracentrifuge tube and ii)centrifuging said sample in said tube to concentrate said viruses, saidultracentrifuge tube comprising an upper region, a middle region and alower region, wherein said centrifuge tube comprises a linearlycontinuous inner surface at least partially defined by said upper,middle and lower regions, wherein an inner diameter of said upper regionis larger than an inner diameter of said middle region and wherein aninner diameter of said middle region is larger than an inner diameter ofsaid lower region and wherein said lower region has a closed bottom, anddetecting the bound dyes in a band of virus, whereby the type of nucleicacid present in the viruses is determined.
 18. The ultracentrifuge tubeof claim 17, wherein said middle region comprises one or moreserrations.
 19. The ultracentrifuge tube of claim 17, wherein said lowerregion has an inner diameter small enough to trap an air bubble betweentwo layers of liquid such that the air bubble will keep said two layersof liquid separate so long as said centrifuge tube is at rest.
 20. Themethod of claim 17, wherein said dyes are added to said sample.
 21. Themethod of claim 17, wherein such dyes are added to a layer of fluidadded to the tube before the addition of said sample and said virusescontact said dyes during centrifugation.
 22. The method of claim 17,wherein said dyes are fluorescent and said bound fluorescent dyes aredetected by passing an exciting fluorescent light through said band ofvirus and determining a wavelength of peak intensity of emittedfluorescent light from said band of virus.
 23. The method of claim 22,which further comprises removing unbound dyes from said tube prior todetermining said wavelength of peak intensity.
 24. The method of claim17, wherein said bound dyes are detected by passing an exciting lightthrough said band of virus and determining the spectral distribution ofthe emitted light.
 25. A method for determining an infectious agenttitre in a biological sample, which comprises measuring the intensity ofemitted fluorescent light of claim
 22. 26. A method for distinguishingbetween single-stranded DNA viruses, double-stranded DNA viruses and RNAviruses present in a biological sample containing viruses, whichcomprises contacting dyes which can distinguish between said viruseswith viruses in said sample, concentrating said viruses by i) adding asample containing said viruses to an ultracentrifuge tube and ii)centrifuging said sample in said tube to concentrate said viruses, saidultracentrifuge tube comprising an upper region, a middle region and alower region wherein an inner diameter of said upper region is largerthan an inner diameter of said middle region and wherein an innerdiameter of said middle region is larger than an inner diameter of saidlower region, wherein said upper region is separated from said middleregion by a first tapering region having a decreasing diameter from saidupper region toward said middle region, and said middle region isseparated from said lower region by a second tapering region having adecreasing diameter from said middle region toward said lower region,and detecting the bound dyes in a band of virus, whereby the type ofnucleic acid present in the viruses is determined.
 27. Theultracentrifuge tube of claim 26, wherein said middle region comprisesone or more serrations.
 28. The ultracentrifuge tube of claim 26,wherein said lower region has an inner diameter small enough to trap anair bubble between two layers of liquid such that the air bubble willkeep said two layers of liquid separate so long as said centrifuge tubeis at rest.
 29. The method of claim 26, wherein said dyes are added tosaid sample.
 30. The method of claim 26, wherein such dyes are added toa layer of fluid added to the tube before the addition of said sampleand said viruses contact said dyes during centrifugation.
 31. The methodof claim 26, wherein said dyes are fluorescent and said boundfluorescent dyes are detected by passing an exciting fluorescent lightthrough said band of virus and determining a wavelength of peakintensity of emitted fluorescent light from said band of virus.
 32. Themethod of claim 31, which further comprises removing unbound dyes fromsaid tube prior to determining said wavelength of peak intensity. 33.The method of claim 26, wherein said bound dyes are detected by passingan exciting light through said band of virus and determining thespectral distribution of the emitted light.
 34. A method for determiningan infectious agent titre in a biological sample, which comprisesmeasuring the intensity of emitted fluorescent light of claim
 31. 35. Amethod of determining which microorganism is present in a biologicalsample which contains a microorganism wherein said method comprises thesteps of: (a) concentrating said microorganism according to yieldconcentrated microorganism by i) adding a sample containing saidmicroorganism to an ultracentrifuge tube and ii) centrifuging saidsample in said tube to concentrate said microorganism, saidultracentrifuge tube comprising an upper region, a middle region and alower region, wherein an inner diameter of said upper region is largerthan an inner diameter of said lower region, wherein said upper regionis separated from said lower region by said middle region having adecreasing diameter from said upper region toward said lower region andwherein said lower region has a closed bottom; (b) extracting nucleicacid from said concentrated microorganism; (c) incubating said nucleicacid with restriction enzymes to produce nucleic acid fragments; (d)staining said nucleic acid or nucleic acid fragments; (e) determining apattern of sizes of said nucleic acid fragments; and (f) comparing saidpattern of sizes with patterns of sizes of nucleic acids, digested withsaid restriction enzymes, obtained from known microorganisms, whereinsaid microorganism in said biological sample is identified as amicroorganism which has an identical restriction fragment pattern. 36.The ultracentrifuge tube of claim 35, wherein said middle regioncomprises one or more serrations.
 37. The ultracentrifuge tube of claim35, wherein said lower region has an inner diameter small enough to trapan air bubble between two layers of liquid such that the air bubble willkeep said two layers of liquid separate so long as said centrifuge tubeis at rest.
 38. The method of claim 35, wherein said sizes of nucleicacid molecules or fragments thereof are determined using flow cytometry.39. The method of claim 35, wherein said sizes of nucleic acid moleculesor fragments thereof are determined by gel electrophoresis.
 40. Themethod of claim 35, wherein said sizes of nucleic acid molecules orfragments thereof are determined by mass spectrometry.
 41. The method ofclaim 35, wherein said size of nucleic acid molecules or fragmentsthereof are determined by optical mapping.
 42. A method of determiningwhich microorganism is present in a biological sample which contains amicroorganism wherein said method comprises the steps of: (a)concentrating said microorganism according to yield concentratedmicroorganism by i) adding a sample containing said microorganism to anultracentrifuge tube and ii) centrifuging said sample in said tube toconcentrate said microorganism, said ultracentrifuge tube comprising anupper region, a middle region and a lower region wherein an innerdiameter of said upper region is larger than an inner diameter of saidmiddle region, wherein (i) an inner diameter of said middle region islarger than an inner diameter of said lower region or (ii) the innerdiameter of said middle region is the same as the inner diameter of saidlower region, wherein that inner diameter is small enough to trap an airbubble between two layers of aqueous liquid such that the air bubblewill keep said two layers of aqueous liquid separate so long as saidcentrifuge tube is at rest, and wherein said lower region has a closedbottom; (b) extracting nucleic acid from said concentratedmicroorganism; (c) incubating said nucleic acid with restriction enzymesto produce nucleic acid fragments; (d) staining said nucleic acid ornucleic acid fragments; (e) determining a pattern of sizes of saidnucleic acid fragments; and (f) comparing said pattern of sizes withpatterns of sizes of nucleic acids, digested with said restrictionenzymes, obtained from known microorganisms, wherein said microorganismin said biological sample is identified as a microorganism which has anidentical restriction fragment pattern.
 43. The method of claim 42,wherein said sizes of nucleic acid molecules or fragments thereof aredetermined using flow cytometry.
 44. The method of claim 42, whereinsaid sizes of nucleic acid molecules or fragments thereof are determinedby gel electrophoresis.
 45. The method of claim 42, wherein said sizesof nucleic acid molecules or fragments thereof are determined by massspectrometry.
 46. The method of claim 42, wherein said size of nucleicacid molecules or fragments thereof are determined by optical mapping.47. A method of determining which microorganism is present in abiological sample which contains a microorganism wherein said methodcomprises the steps of: (a) concentrating said microorganism accordingto yield concentrated microorganism by i) adding a sample containingsaid microorganism to an ultracentrifuge tube and ii) centrifuging saidsample in said tube to concentrate said microorganism, saidultracentrifuge tube comprising an upper region, a middle region and alower region, wherein said centrifuge tube comprises a linearlycontinuous inner surface at least partially defined by said upper,middle and lower regions, wherein an inner diameter of said upper regionis larger than an inner diameter of said middle region and wherein aninner diameter of said middle region is larger than an inner diameter ofsaid lower region and wherein said lower region has a closed bottom; (b)extracting nucleic acid from said concentrated microorganism; (c)incubating said nucleic acid with restriction enzymes to produce nucleicacid fragments; (d) staining said nucleic acid or nucleic acidfragments; (e) determining a pattern of sizes of said nucleic acidfragments; and (f) comparing said pattern of sizes with patterns ofsizes of nucleic acids, digested with said restriction enzymes, obtainedfrom known microorganisms, wherein said microorganism in said biologicalsample is identified as a microorganism which has an identicalrestriction fragment pattern.
 48. The ultracentrifuge tube of claim 47,wherein said middle region comprises one or more serrations.
 49. Theultracentrifuge tube of claim 47, wherein said lower region has an innerdiameter small enough to trap an air bubble between two layers of liquidsuch that the air bubble will keep said two layers of liquid separate solong as said centrifuge tube is at rest.
 50. The method of claim 47,wherein said sizes of nucleic acid molecules or fragments thereof aredetermined using flow cytometry.
 51. The method of claim 47, whereinsaid sizes of nucleic acid molecules or fragments thereof are determinedby gel electrophoresis.
 52. The method of claim 47, wherein said sizesof nucleic acid molecules or fragments thereof are determined by massspectrometry.
 53. The method of claim 47, wherein said size of nucleicacid molecules or fragments thereof are determined by optical mapping.54. A method of determining which microorganism is present in abiological sample which contains a microorganism wherein said methodcomprises the steps of: (a) concentrating said microorganism accordingto yield concentrated microorganism by i) adding a sample containingsaid microorganism to an ultracentrifuge tube and ii) centrifuging saidsample in said tube to concentrate said microorganism, saidultracentrifuge tube comprising an upper region, a middle region and alower region wherein an inner diameter of said upper region is largerthan an inner diameter of said middle region and wherein an innerdiameter of said middle region is larger than an inner diameter of saidlower region, wherein said upper region is separated from said middleregion by a first tapering region having a decreasing diameter from saidupper region toward said middle region, and said middle region isseparated from said lower region by a second tapering region having adecreasing diameter from said middle region toward said lower region;(b) extracting nucleic acid from said concentrated microorganism; (c)incubating said nucleic acid with restriction enzymes to produce nucleicacid fragments; (d) staining said nucleic acid or nucleic acidfragments; (e) determining a pattern of sizes of said nucleic acidfragments; and (f) comparing said pattern of sizes with patterns ofsizes of nucleic acids, digested with said restriction enzymes, obtainedfrom known microorganisms, wherein said microorganism in said biologicalsample is identified as a microorganism which has an identicalrestriction fragment pattern.
 55. The ultracentrifuge tube of claim 54,wherein said middle region comprises one or more serrations.
 56. Theultracentrifuge tube of claim 54, wherein said lower region has an innerdiameter small enough to trap an air bubble between two layers of liquidsuch that the air bubble will keep said two layers of liquid separate solong as said centrifuge tube is at rest.
 57. The method of claim 54,wherein said sizes of nucleic acid molecules or fragments thereof aredetermined using flow cytometry.
 58. The method of claim 54, whereinsaid sizes of nucleic acid molecules or fragments thereof are determinedby gel electrophoresis.
 59. The method of claim 54, wherein said sizesof nucleic acid molecules or fragments thereof are determined by massspectrometry.
 60. The method of claim 54, wherein said size of nucleicacid molecules or fragments thereof are determined by optical mapping.61. A method of identifying a microorganism in a biological sample,wherein said method comprises the steps of: (a)concentrating saidmicroorganism by i) adding a sample containing said microorganism to anultracentrifuge tube and ii) centrifuging said sample in said tube toconcentrate said microorganism, said ultracentrifuge tube comprising anupper region, a middle region and a lower region, wherein an innerdiameter of said upper region is larger than an inner diameter of saidlower region, wherein said upper region is separated from said lowerregion by said middle region having a decreasing diameter from saidupper region toward said lower region and wherein said lower region hasa closed bottom; and (b) incubating with antibodies specific for knownmicroorganisms, wherein if said antibodies bind to said concentratedmicroorganism then said microorganism is identified as the microorganismto which the antibodies are known to bind, by their fluorescence. 62.The ultracentrifuge tube of claim 61, wherein said middle regioncomprises one or more serrations.
 63. The ultracentrifuge tube of claim61, wherein said lower region has an inner diameter small enough to trapan air bubble between two layers of liquid such that the air bubble willkeep said two layers of liquid separate so long as said centrifuge tubeis at rest.
 64. The method of claim 61, wherein said fluorescentantibodies are present in said upper region of said centrifuge tubeduring centrifuigation of said biological sample, attach to themicroorganism for which they are specific during incubation, cosedimentand coband with said microorganism, and are detected by the fluorescenceof said antibody-microorganism conjugate band.
 65. The method of claim64, wherein a plurality of species of antibody is present in said upperregion of said centrifuge tube during centrifugation of said biologicalsample and wherein each species of antibody is labeled with a markerdistinct from any marker on any other species of antibody present insaid upper region.
 66. The method of claim 61, in which the antibodymicroorganism complex has a banding density different from that of thefree microorganism, thus allowing the presence of the complex to bedetected.
 67. A method of identifying a microorganism in a biologicalsample, wherein said method comprises the steps of: (a)concentratingsaid microorganism by i) adding a sample containing said microorganismto an ultracentrifuge tube and ii) centrifuging said sample in said tubeto concentrate said microorganism, said ultracentrifuge tube comprisingan upper region, a middle region and a lower region wherein an innerdiameter of said upper region is larger than an inner diameter of saidmiddle region, wherein (i) an inner diameter of said middle region islarger than an inner diameter of said lower region or (ii) the innerdiameter of said middle region is the same as the inner diameter of saidlower region, wherein that inner diameter is small enough to trap an airbubble between two layers of aqueous liquid such that the air bubblewill keep said two layers of aqueous liquid separate so long as saidcentrifuge tube is at rest, and wherein said lower region has a closedbottom; and (b) incubating with antibodies specific for knownmicroorganisms, wherein if said antibodies bind to said concentratedmicroorganism then said microorganism is identified as the microorganismto which the antibodies are known to bind, by their fluorescence. 68.The method of claim 67, wherein said fluorescent antibodies are presentin said upper region of said centrifuge tube during centrifugation ofsaid biological sample, attach to the microorganism for which they arespecific during incubation, cosediment and coband with saidmicroorganism, and are detected by the fluorescence of saidantibody-microorganism conjugate band.
 69. The method of claim 68,wherein a plurality of species of antibody is present in said upperregion of said centrifuge tube during centrifugation of said biologicalsample and wherein each species of antibody is labeled with a markerdistinct from any marker on any other species of antibody present insaid upper region.
 70. The method of claim 67, in which the antibodymicroorganism complex has a banding density different from that of thefree microorganism, thus allowing the presence of the complex to bedetected.
 71. A method of identifying a microorganism in a biologicalsample, wherein said method comprises the steps of: (a)concentratingsaid microorganism by i) adding a sample containing said microorganismto an ultracentrifuge tube and ii) centrifuging said sample in said tubeto concentrate said microorganism, said ultracentrifuge tube comprisingan upper region, a middle region and a lower region, wherein saidcentrifuge tube comprises a linearly continuous inner surface at leastpartially defined by said upper, middle and lower regions, wherein aninner diameter of said upper region is larger than an inner diameter ofsaid middle region and wherein an inner diameter of said middle regionis larger than an inner diameter of said lower region and wherein saidlower region has a closed bottom; and (b) incubating with antibodiesspecific for known microorganisms, wherein if said antibodies bind tosaid concentrated microorganism then said microorganism is identified asthe microorganism to which the antibodies are known to bind, by theirfluorescence.
 72. The ultracentrifuge tube of claim 71, wherein saidmiddle region comprises one or more serrations.
 73. The ultracentifugetube of claim 71, wherein said lower region has an inner diameter smallenough to trap an air bubble between two layers of liquid such that theair bubble will keep said two layers of liquid separate so long as saidcentrifuge tube is at rest.
 74. The method of claim 71, wherein saidfluorescent antibodies are present in said upper region of saidcentrifuge tube during centrifugation of said biological sample, attachto the microorganism for which they are specific during incubation,cosediment and coband with said microorganism, and are detected by thefluorescence of said antibody-microorganism conjugate band.
 75. Themethod of claim 74, wherein a plurality of species of antibody ispresent in said upper region of said centrifuge tube duringcentrifugation of said biological sample and wherein each species ofantibody is labeled with a marker distinct from any marker on any otherspecies of antibody present in said upper region.
 76. The method ofclaim 71, in which the antibody microorganism complex has a bandingdensity different from that of the free microorganism, thus allowing thepresence of the complex to be detected.
 77. A method of identifying amicroorganism in a biological sample, wherein said method comprises thesteps of: (a)concentrating said microorganism by i) adding a samplecontaining said microorganism to an ultracentrifuge tube and ii)centrifuging said sample in said tube to concentrate said microorganism,said ultracentrifuge tube comprising an upper region, a middle regionand a lower region wherein an inner diameter of said upper region islarger than an inner diameter of said middle region and wherein an innerdiameter of said middle region is larger than an inner diameter of saidlower region, wherein said upper region is separated from said middleregion by a first tapering region having a decreasing diameter from saidupper region toward said middle region, and said middle region isseparated from said lower region by a second tapering region having adecreasing diameter from said middle region toward said lower region;and (b) incubating with antibodies specific for known microorganisms,wherein if said antibodies bind to said concentrated microorganism thensaid microorganism is identified as the microorganism to which theantibodies are known to bind, by their fluorescence.
 78. Theultracentrifuge tube of claim 77, wherein said middle region comprisesone or more serrations.
 79. The ultracentifuge tube of claim 77, whereinsaid lower region has an inner diameter small enough to trap an airbubble between two layers of liquid such that the air bubble will keepsaid two layers of liquid separate so long as said centrifuge tube is atrest.
 80. The method of claim 77, wherein said fluorescent antibodiesare present in said upper region of said centrifuge tube duringcentrifugation of said biological sample, attach to the microorganismfor which they are specific during incubation, cosediment and cobandwith said microorganism, and are detected by the fluorescence of saidantibody-microorganism conjugate band.
 81. The method of claim 80,wherein a plurality of species of antibody is present in said upperregion of said centrifuge tube during centrifugation of said biologicalsample and wherein each species of antibody is labeled with a markerdistinct from any marker on any other species of antibody present insaid upper region.
 82. The method of claim 77, in which the antibodymicroorganism complex has a banding density different from that of thefree microorganism, thus allowing the presence of the complex to bedetected.